[Histonet] TRAP and ALP staining

[Mari Ferrarini]

Hello all,

 

I'm currently trying to do TRAP and ALP staining in MMA (Technovit 9100) and GMA (cold embedding) embedded sections.

The thing is I don't have a lot of experience and don't really know what could go wrong.

I'm doing the stainings proposed by Chappard and Liu for TRAP and the staining proposed by Liu for ALP, as follows:

 

TRAP LIU:

(1 mL : 1 mL) 4% NaNO2 + 4% Pararosaniline;

23 mL 0,1M Acetate Buffer;

8 mg Naphtol AS-TR Phosphate

0,3 mL Dimethylformamide;

57,5 mg Sodium Tartrate

pH: 5; 37oC/1 h;

 

ALP LIU:

24 mg Fast Blue BB

30 mL 0,1M Tris Buffer;

8 mg naphtol AS-TR Phosphate

0,3 mL Dimethylformamide

10 mg MgCl2

pH: 9; 37oC/30 min;

 

TRAP CHAPPARD:

100 mg Sodium alfa-naphtyl phosphate

200 mg Fast Violet B;

15 mg Sodium Tartrate;

100 mL 0,1M Acetate Buffer, pH 5;

37oC/30 min

 

Is there a problem if I change the phosphate? I only have a naphthol AS-MX phosphate. Is it equivalent for these procedures?

I'm not really sure either on the kind of mounting media I'm supposed to use. Aqueous mounting for both MMA and GMA?

I'm also not sure of how to attach the sections to the slides prior to staining in a way that doesn't affect much the enzyme activity.

 

Thank you very much for the time and patience, any help would be appreciated.

 

Sincerely,

 

Mariana Ferrarini

Lyon, France.

 

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